Milk is a rich source of essential nutrients such as calcium, protein, and vitamins, which are crucial for the growth and development of the organism. Antibiotics in milk are a significant concern because they can contribute to the development of antibiotic-resistant bacteria and pose health risks to consumers. Residues of antibiotics in milk can occur due to improper use of medications in dairy cows or failure to observe the required withdrawal periods before milking.The regulation of antibiotic levels in milk in the Republic of Croatia is governed by several laws and regulations, including the Food Act, the Veterinary Act, the Regulation on Maximum Residue Levels of Pesticides, and the Act on Veterinary-Medical Products. The Ministry of Agriculture, HAPIH, and the Veterinary Inspection are key bodies responsible for overseeing the implementation of these laws. We have addressed various aspects of antibiotic use in the dairy industry, including specific types of antibiotics such as tetracyclines, cephalosporins, and beta-lactam antibiotics, their mechanisms of action, and their use in treating cattle. The goal of testing milk samples for the presence of antibiotics is to ensure the milk’s safety and protect consumers from potential harmful residues. The TwinSensor Plus device is used for the rapid and accurate detection of antibiotics in milk samples, ensuring the quality and safety of dairy products. Negative results for antibiotic presence in milk confirm its safety and compliance with established quality standards. In conclusion, regular testing of milk for antibiotics is crucial for maintaining the safety of the food chain and consumer trust.